Following Molecular Transitions with Single Residue Spatial and Millisecond Time Resolution

Inna Shcherbakova, Somdeb Mitra, Robert H. Beer, Michael D. Brenowitz

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

"Footprinting" describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions, respectively. The hydroxyl radical (•OH) is a uniquely insightful footprinting probe by virtue of it being among the most reactive chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved •OH footprinting is presented based on the Fenton reaction, Fe(II) + H2O2 → Fe(III) + •OH + OH-. It is implemented using a standard three-syringe quench-flow mixer. The utility of this method is demonstrated by its application to the studies on RNA folding. Its applicability to a broad range of biological questions involving the function of DNA, RNA, and proteins is discussed.

Original languageEnglish (US)
Pages (from-to)589-615
Number of pages27
JournalMethods in Cell Biology
Volume84
DOIs
StatePublished - 2008

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RNA Folding
Syringes
Thermodynamics
Oxidants
Hydroxyl Radical
Nucleic Acids
Catalytic Domain
Polymers
Proteins
RNA
Ligands
DNA
hydroxide ion

ASJC Scopus subject areas

  • Cell Biology

Cite this

Following Molecular Transitions with Single Residue Spatial and Millisecond Time Resolution. / Shcherbakova, Inna; Mitra, Somdeb; Beer, Robert H.; Brenowitz, Michael D.

In: Methods in Cell Biology, Vol. 84, 2008, p. 589-615.

Research output: Contribution to journalArticle

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